Project Summary The proposed research will interrogate the natural product scaffold of carolacton as a starting point for agents that perturb S. mutans (SMU) biofilms. Carolacton is remarkable: instead of dispersing established biofilms or killing planktonic cells, carolacton targets cells transitioning into the biofilm state, and it is effective at very low concentrations. Here we present a multi-faceted approach, including organic synthesis, molecular genetics, proteomics, transcriptomics, and microbiological assays that begins with carolacton but has as an overarching goal the development of tool compounds that can be used to understand SMU biofilm processes within a multispecies environment. The first specific aim seeks to identify the specific target(s) of carolacton and a recently discovered analog that also has profound effects on SMU biofilms. This approach will employ genetic, transcriptomic, and MS-proteomic techniques to identify candidates that bind carolacton, from which we will confirm the targets using biochemical studies. This will shed light on the processes that the natural product affects and allow for a broader evaluation of the target in general biofilm processes. The second specific aim involves the chemical synthesis of rationally designed compounds related to carolacton. Proposed compounds will focus on four main aspects: 1) obtaining a detailed structure- activity relationship understanding of the carolacton structure; 2) determining the minimally complex scaffold that retains biological activity; 3) improving the physiochemical properties; and 4) identification of a lead compound for use in dental composites. Central to the efficient and concise strategies proposed is the knowledge gained in our previously described total synthesis. The third aim will investigate the biological properties of the tool compounds both in single species and multispecies biofilms. Preliminary results have identified that both carolacton and a newly synthesized analog cause a unique and specific phenotypic response to S. mutans biofilms. Therefore, all analogs will be tested against both planktonic and biofilm cells with S. mutans UA159 using confocal microscopy. Lead compounds will then be analyzed in assays with clinical isolates of both S. mutans and other bacteria involved in dental caries in collaboration with the Univ. of Rochester and the Univ. of Florida. Concurrently, compounds will be evaluated in mixed-species biofilms between S. mutans and commensal bacteria (i.e. S. sanguinis, S. gordonii) to identify pathogen-specific inhibitors. Previous work has demonstrated that oral pathogens with compromised signaling systems are reduced viability in the presence of commensal bacteria and we intend to build on this earlier work.